Human respiratory syncytial virus (HRSV) is a major cause of severe lower respiratory tract disease in infants and children worldwide as well as in immunosuppressed individuals and the elderly. Amidst ongoing efforts to develop human respiratory syncytial virus specific vaccines and therapeutic agents, prevention and treatment of human respiratory syncytial virus disease remain a significant challenge. Human respiratory syncytial virus is the type species of the genus Pneumovirus within the family Paramyxoviridae and contains a negative-sense, single-stranded RNA genome that expresses eleven known proteins from ten genes. Three proteins, SH (small hydrophobic), G (attachment), and F (fusion), have been characterized as transmembrane glycoproteins and are detected in purified virions. On the surface of infected cells, the G and F proteins concentrate in cell-associated, virus-induced filamentous structures with variable lengths of up to 10 μm. The G and F proteins contain the major antigenic epitopes of human respiratory syncytial virus, and their roles in the anti-HRSV imnune response have been investigated extensively.
The SH protein is a small integral membrane protein of unknown function, with a relatively low amino acid conservation among human respiratory syncytial virus strains. Previous studies indicate that SH is dispensable for human respiratory syncytial virus growth in cell culture, and its absence has little impact on the ability of the virus to replicate in the respiratory tracts of mice and chimpanzees.
The G gene of human respiratory syncytial virus expresses both a type II membrane-anchored glycoprotein and a soluble protein. G protein is heavily O-glycosylated and shows significant structural similarities to mucinous proteins. The G protein was initially characterized as providing an attachment function, and domains in G have since been identified that bind to sulfated glycosaminoglycans on the cell surface in vitro. The requirement for G protein in infectivity in cell culture varies depending on the cell type. Both a cold-adapted virus in which most of the sequence encoding the SH and G proteins is absent, and an engineered virus lacking the G gene replicate efficiently in Vero cells. However, replication of these G-deleted viruses is significantly impaired in HEp-2 cells as well as in mice, cotton rats, and humans.
The fusion protein, F, is a type I transmembrane glycoprotein that mediates the formation of syncytia typically observed in human respiratory syncytial virus infected cells. F protein is thought to direct entry of human respiratory syncytial virus at the plasma membrane in a pH-independent manner. Among the transmembrane glycoproteins, F appears to be a critical component for virus transmission, as F, matrix (M) protein, and the nucleocapsid were found to be the minimal requirements for production of infectious particles, and viruses that express F as the only glycoprotein propagated efficiently in Vero cells.
A previous attempt to vaccinate young children against respiratory syncytial virus (RSV) employed a parenterally administered formalin-inactivated RSV vaccine. Unfortumately, administration of this vaccine in several field trials was shown to be associated with the development of a significantly exacerbated illness following subsequent natural infection with RSV.
Following the lack of success with the formalin-inactivated RSV vaccine, emphasis was placed on the development of live attenuated vaccines. For example, cold adaptation, a process by which virus is adapted to grow at temperatures colder than those at which it normally optimally grows, has been used to develop temperature sensitive, attenuated RSV mutants for consideration as vaccines (Maassab, et al., VACCINE, 3:355-369 (1985)).
In most cases, the attenuated RSV, like the wild-type RSV, is unstable above 0° C. The attempt to store RSV stocks below 0° C. is also unsuccessful, partially because of the sensitivity of RSV to freezing and thawing. Moreover, storing virus stocks under 0° C. requires elaborate and expensive cold chain equipment, which significantly increases the cost associated with the use of RSV as a vaccine. Furthermore, the instability of RSV presents a challenge for producing high-titer RSV vaccines.